Surgery and trauma conditions often require blood transfusion. Blood substitutes are needed for use in emergency transfusions under conditions of extreme blood loss. PEGylated hemoglobins are hemoglobins that that have been chemically modified through the attachment of polyethyleneglycol (PEG) to the protein. The mode of attachment, the site(s) of attachment, and the size and number of attached PEG chains can be varied (e.g., U.S. Pat. Nos. 5,585,484, 5,750,725, and 6,017,943; U.S. Patent Application Publication Nos. 2003/0187248 A1 and 2004/0002443 A1). PEGylated hemoglobins show ever increasing promise as blood substitutes for emergency use. An important issue, both economic and clinical, is the development of protocols to store these materials for long periods without loss of efficacy. This point is especially significant since the production, transportation and storage costs for these materials are likely to be high. The production of a reconstitutable powdered form of a blood substitute that allows for long term storage and facile transport at ambient temperatures would significantly reduce costs and increase the clinical applicability of these materials under emergency and combat situations.
Long term storage of hemoglobins is typically complicated by autoxidation (formation of methemoglobin), whereby the ferrous (reduced) oxygen carrying form of the protein converts to the ferric (oxidized) non-oxygen transporting form referred to as methemoglobin. In addition to being ineffective as an oxygen carrier, the met form of hemoglobin has an increased tendency to release the heme portion of hemoglobin. The heme is toxic when free of the protein.
Liquid forms of hemoglobin including PEGylated hemoglobins can be prepared and stored for long periods at cryogenic temperatures with minimal formation of met hemoglobin. The downside to this approach is the cost of maintaining the product at cryogenic temperatures, the cost of shipping the inherently heavy solutions (frozen or liquid) and the ability to maintain these samples for extended periods in emergency vehicles especially under combat conditions. Far more preferable would be a dry stable (with respect to methemoglobin formation) light-weight product that can be stored at higher temperatures, transported for long stretches without the need for ongoing refrigeration and easily reconstituted in the field.
There are two commonly used methods for producing powdered forms of protein. The more routine approach is freeze-drying or lyophilization. Lyophilization protocols consist of first freezing the liquid sample and then drying it under a vacuum to remove water. The other approach, based on air drying, utilizes a special nozzle technology to produce a high speed jet of material that forms easily dried micro-droplets.
Successful lyophilization requires that proteins survive both the freezing and drying phases of the process. Many proteins including hemoglobin undergo some degree of denaturation when lyophilized from an aqueous solution. The addition of simple (non-reducing) sugars especially glass forming sugars such as trehalose has been shown to greatly limit protein damage during lyophilization. Unfortunately, the combination of trehalose and PEG typically results in phase separation and loss of protection during lyophilization. Furthermore, lyophilization of oxygenated derivatives of PEGylated hemoglobins using trehalose results in dried material almost completely in the met form. The met formation also occurs when trehalose-containing samples of PEGylated oxygenated hemoglobin are allowed to air dry without resorting to lyophilization.